Characterization of potential insertion sites in the core antigen of hepatitis B virus by the use of a short-sized model epitope

Core particles of hepatitis B virus (HBV) are able to improve the immunogen icity of foreign sequences exposed on the particle surface. The insertion s ite in the core antigen of HBV (HBcAg) determines the surface presentation and thus the immunogenicity of the foreign sequence. For direct comparison of the value of potential insertion sites in the core antigen, we construct ed vectors allowing insertions of a model marker epitope DPAFR. This epitop e was inserted at the N-terminus, the c/e1 loop, behind amino acid (aa) 144 and behind aa 183 (DPAF only). In addition, we generated a mosaic construc t allowing the co-expression of HBcAg a nd a HBcAg/ DPAFR fusion protein du e to a suppressor tRNA-mediated readthrough mechanism. All 6 constructs all owed the formation of chimaeric or mosaic core-like particles. Western blot analyses and a direct ELISA demonstrated the presence of the DPAFR sequenc e in the chimaeric and mosaic particles. Competitive ELISA and immune elect ron-microscopic data suggested the c/e1 loop as the insertion site of choic e for presenting foreign sequences on the surface of chimaeric HBV core par ticles. However, the N-terminal fusion also allowed partial surface exposur e of the DPAFR motif, In contrast, in particles of constructs carrying the DPAFR insert at aa position 144 or 183, respectively, the epitope seemed no t to be surface accessible.